Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine

Background There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. Methods The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). Results None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. Conclusions This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. Trial registration Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369; date: 06 June 2017). Supplementary Information The online version contains supplementary material available at 10.1186/s12916-021-02150-x.

Single Nucleotide Variants (SNVs) and small insertions and deletions were called with two different software tools, VarScan somatic version 2.3 [33], and SNVer version 0.5.3 [36]. Calls were filtered requiring minimum-read-depth = 10, alternate allele frequency in strain (AF) > 0.25, and discarding events near telomeres (defined as first and last 10% of each chromosome).
Static adhesion assay 2.5 µL purified ICAM-1 and CD36 at 50 µg/mL and a PBS control were spotted in triplicate onto a 60 mm petri dish and incubated in a humidified container for 1 hour at 37°C. The protein was removed and the dish incubated for 1 hour in blocking solution (PBS/1% BSA) at 37°C. An IE suspension of 2% parasitaemia and 1.5% haematocrit in binding buffer (RPMI 1640 with 25 mM HEPES, 11 mM glucose, 2 mM glutamine, pH 7.2) was added and incubated at 37°C for 1 h with gentle resuspension every 10 minutes. Unbound IE were removed by washing with binding buffer and the bound IE fixed in 1% glutaraldehyde, stained with Giemsa, counted by microscopy and the mean IE per mm 2 surface calculated. The mean of the triplicates was calculated and non-specific binding to PBS subtracted to determine receptor specific binding in 4 independent experiments.

Flow adhesion assay
The assay was performed using primary Human Dermal Microvascular Endothelial Cells (HDMEC, Promocell, Germany) and the Cellix microfluidics system (https://www.wearecellix.com/) as previously described [38]. Briefly, HDMEC were cultured as per manufacturer's instructions and used up to passage 7. For the assay, cells were detached with Accutase ® and seeded in Vena8 biochips (Cellix) coated with Attachment Factor. Medium was changed every hour and after 4-5 hours, when cells formed a confluent monolayer, an IE suspension of 2% parasitaemia and 5% haematocrit in binding buffer was flowed through at shear stress of 0.4 dyne/cm 2 for 5 minutes at 37°C. After a wash with binding buffer, the bound IE were counted in 15 fields by microscopy and the mean IE per mm 2 HDMEC surface calculated. Four independent experiments were performed and in two experiments HDMEC were stimulated overnight with 10 ng/mL TNF.

Text S3: Methodology for scanning electron microscopy
Parasite infected red blood cells were fixed with 0.05% glutaraldehyde for 25 minutes in PBS at room temperature and further in 2.5% glutaraldehyde for 1.5 hrs [39]. Fixed cells were washed in H2O three times and subsequently adhered to poly-l-lysine coated glass coverslips for 5 minutes. Coverslips with bound cells were sequentially washed for 5 min each in 20%, 50%, 70%, 80%, 90%, 95% and 100% (×3) ethanol before transfer to a Lecia CPD300 critical point dryer, run for 24 fill cycles. Coverslips were gold-coated for 75 s at 25 mA using a Dynavac sputter coating instrument with rotating mount. The coating thickness was measured at ~0.3 nm on the internal quartz crystal microbalance. Images were acquired using an FEI Teneo SEM using the ETD detector in Optiplan mode, at a working distance of 5 mm, a beam current of 50 pA and a 2 kV accelerating voltage.

Text S4: Methodology for var gene expression analysis
Parasites were cultivated in vitro at 3% haematocrit for the duration specified in Table S1 below prior to harvesting infected erythrocytes from 60 mL of ring stage culture. Parasites were dissolved in 11 mL of trizol (Invitrogen) and RNA was extracted and purified using RNeasy mini columns (Qiagen) [43]. Contaminating DNA was digested with DNaseI (Qiagen) and digestion assessed by qPCR prior to another round of RNA purification (Qiagen RNeasy mini). RNA quality was assessed by Agilent bioAnalyser (RIN: 7.5-9.5). The harvested RNA was reverse transcribed and used to determine the var gene transcriptional profile by qRT-PCR using SYBR green master mix (Applied Biosystems) using published protocols [41] and primers [40]. Data were analysed as 2 -(ΔΔCt) using the skeleton binding protein 1 (SBP1) gene as the normalising control [42] and P. falciparum 3D7 gDNA as the calibrator. No copy number differences were seen between the subject samples and the control sample. Structural variant (SV) analysis found the kahrp gene knockout in all three samples. The only new SV was a rearrangement in the sample from subject 5, affecting the first PfEMP1 var gene at the start of both chromosome 7 and chromosome 10. A small number of low-quality small variants (SNVs and indels) were observed, but no non-synonymous SNVs or indels in genes of known function.

Text S6: Results of scanning electron microscopy analysis of parasite-infected erythrocytes
Scanning electron microscopy (SEM) was performed to determine if breakthrough parasites had reverted to a wild-type knob phenotype. A reference image of wild-type 3D7 trophozoiteinfected RBC is presented in Figure 4A showing abundant knobs on the external surface of the wild-type 3D7 parasite-infected RBCs. In contrast to this, no knobs are observed on the external surface of the kahrp-parasites prior to inoculation ( Figure 4B). The ex vivo samples from subject 5 and 6 were imaged and show no knobs at the external surface (Figure 4 C,D). These results show that the breakthrough parasites imaged from subject 5 and 6 have not reverted to wild-type and present with the same knob-minus phenotype as the kahrp-parasite pre-inoculation.

Text S7. Results of cytoadherence assays
As shown in Figure 5, the cytoadherence characteristics of IE isolated from subject 5 and 6 are not significantly altered compared to 3D7 kahrp-IE. No binding to purified ICAM-1 ( Figure 5A) and modest binding to purified CD36 ( Figure 5B) could be detected under static binding conditions. There was no binding to HDMEC under flow conditions for 3D7 kahrp-IE and subject 6 IE, and the small amount of binding for subject 5 IE was not significantly different ( Figure 5C). In contrast, the laboratory strain ItG did bind to the purified receptors and HDMEC as previously reported [37], indicating the robustness of the cytoadherence assays.

Text S8: Results of var gene expression analysis
The var genes most abundantly expressed by parasites from subject 5 and subject 6 had no published associations with virulence that could explain improved adhesion and thus escape of splenic clearance. In subject 5, a broad range of var genes were transcribed with a group B and a group B/A var genes dominating (Table S2 below). The gene PF3D7_0632500 in subject 5 might bind the endothelial cell receptor ICAM 1 via its DBLβ5 domain. This class of domains have been implicated in adhesion to ICAM 1 and cerebral malaria however this specific domain has not been shown to bind ICAM 1.
The most abundant var transcripts in parasites from subject 6 were detected by a single set of primers that cannot discriminate between var genes PF3D7_1240400 and PF3D7_1240900 (Table S2 below). These two var genes and one of the two most abundant var transcripts in subject 7 all bind the broadly expressed host receptor CD36. Adhesion to CD36 is not associated with severe malaria. The PfEMP1 PF3D7_1240600 expressed by the kahrpparasites used for inoculation also binds CD36, thus the selection of parasites expressing PF3D7_1240400 and/or PF3D7_1240900 in subject 6 was probably not due to selection for adhesion to CD36. Whether the other domains of these PfEMP1s mediate adhesion to other receptors is unknown but could explain the observed selection of a switched parasite population in subject 6.
The level of expression of each gene for the three replicate kahrp-subject 7 samples were averaged and these values grouped with the kahrp-subject 5 and 6 gene expression values as replicates for comparison with the two kahrp-pre inoculation samples as replicates in a multiple t test comparison using the Holm-Sidak method to determine statistical significance in multiple comparisons with alpha=0.05. The expression of the single group C var gene PF3D7_1240600 was significantly reduced in the subjects' samples compared to the pre inoculation samples (p<0.0001). The expression of none of the other var genes differed significantly between the subjects' samples and the pre-inoculation samples. This was unsurprising given the variation between the individual var gene expression patterns between the subjects' samples, although notably subjects 5 and 6 were much more similar to each other than subject 7. If only subjects 5 and 6 were used as replicates to compare to the preinoculation controls then the var genes PF3D7_1240400/PF3D7_1240900 were significantly upregulated in the subjects' samples (p=0.00018) and PF3D7_1240600 was significantly down-regulated (p<0.0001).